Photodynamic therapy (PDT) is a clinically approved treatment based on the administration of a photosensitizing molecule, its accumulation in the target tissue, and then illumination with light selectively absorbed by the photosensitizer. This selectivity is improved using photosensitizers that absorb light in the phototherapeutic window (650-850 nm), where tissues have higher optical penetration depths (e.g., δ=2.3 mm at 750 nm) (1). The absorption of light leaves the photosensitizer in an electronically excited state that reacts with substrate molecules by electron transfer reactions with the formation of superoxide anion and hydroxyl radicals (type I reaction), or to transfer its energy to ground-state molecular oxygen generating singlet oxygen (type II reaction). These photogenerated reactive oxygen species (ROS) trigger biological mechanisms that make PDT an effective anti-cancer procedure (2).
The paradigm of PDT in the treatment of hyperproliferative disorders has been that stable dyes with a stronger absorption of light in the phototherapeutic window and with high ROS quantum yields (ΦROS) should be better photosensitizers. Additionally, the development of PDT photosensitizers targeting Gram-negative bacteria has been guided by the need to have at least one positive charge present in the photosensitizer (3). Such photosensitizers are usually porphyrin derivatives (chlorins or bacteriochlorins) with molecular weights higher than 600 Da. Although photodynamic inactivation of bacteria suspensions by 5-6 orders of magnitude using micromolar photosensitizer concentrations and light doses ca. 10 J/cm2 was achieved (4), the transfer to clinical applications has been unsuccessful. A likely reason for the failure to translate the photodynamic inactivation of bacteria suspensions to clinical settings is the incompatibility between the large size of the photosensitizers that absorb infrared light and the small molecular size required for rapid diffusion in the biofilms and uptake by the bacteria. Similar difficulties have been found in the transfer of PDT photosensitizers to the topical treatment of dermatological disorders. Whereas photosensitizers such as porfimer sodium (trade name Photofrin®) and temoporfin (Foscan®) have obtained approval for cancer indications using intravenous administration, topical applications have not been transferred to the clinical (5). Again, the failure of topical applications of photosensitizers to elicit therapeutic effects is likely related to the difficulty of such photosensitizers to cross the outer layer of the skin, called stratum corneum, and reach their targets. The stratum corneum is the principal barrier to the percutaneous penetration of exogenous molecules.
The best maximum flux (Jmax) of a drug across the skin after topical application is strongly limited by the molecular weight (MW) of the drug (6),log Jmax=−3.90−0.0190 MW
For example, drugs with MW=600 or 700 Da should have Jmax=5×10−16 or 6×10−18 mol/(cm2 h), respectively. These calculations show that a modest increase in the molecular weight above 600 Da can lead to a dramatic decrease in the transdermal flux of the photosensitizers. In practical terms, a photosensitizer of 700 Da is likely to take 100 times longer to reach a therapeutic concentration in a subcutaneous target than a photosensitizer of 600 Da. Another critical property that drugs intended for topical applications must meet, is adequate solubility within the lipid domains of the stratum corneum to permit diffusion through this domain whilst still having sufficient hydrophilic nature to allow partitioning into the viable tissues of the epidermis. Drugs that meet this determinant have the logarithm of their n-octanol-water partition coefficient (log POW) between 1 and 3 (7).
The ideal photosensitizer for topical applications of PDT and for photoinactivation of bacteria must have a molecular weight MW<700 Da, a log POW between 1 and 3, a high molar absorption coefficient ε>30,000 M−1 cm−1 between 650 and 850 nm, and a ROS quantum yield ΦROS>0.3. Additionally, the photostability of the photosensitizer is also critical to the success of PDT (8). The photostability of a photosensitizer can be compared with the turnover of a chemical catalyst: it is related with the number of moles of substrate that a mole of catalyst can convert (i.e., the number of ROS generated) before the catalyst (i.e., photosensitizer) is inactivated (i.e., photodecomposes). The two most widely used photosensitizers for PDT of cancer are porfimer sodium (trade name Photofrin®) and temoporfin (proprietary name Foscan®). Porfimer sodium is a mixture of oligomers formed by ether and ester linkages of up to eight porphyrin units, relatively soluble in aqueous solutions, with log POW≈0. Porfimer sodium is not a single molecular entity and does not have a characteristic molecular weight, but the molecular weight of the smallest dimmer exceeds 1000 Da. Temoporfin is the very lipophilic 5,10,15,20-tetra (m-hydroxyphenyl)chlorin with a molecular weight of 680 Da and log POW=5.5 at physiological pH. The singlet oxygen quantum yields of porfimer sodium and temoporfin are 0.36 and 0.43, respectively (8). Their maximum absorption peaks in the red are at λmax=630 nm with a molar absorption coefficient ε630=1170 M−1 cm−1 for porfimer sodium, and λmax=650 nm with ε650=29600 M−1 cm−1 for temoporfin. They are relatively photostable, with photodecomposition quantum yields Φpd=5.5×10−5 and 3.3×10−5 for porfimer sodium and temoporfin, respectively. When porfimer sodium or temoporfin are incubated with CT26 (mouse colon adenocarcinoma) cells and, after washing, illuminated with laser light of the wavelength matching their red absorption bands to deliver a light dose of 1 J/cm2, it was seen that a porfimer sodium concentration of 18 μM (estimated on the basis of the molecular weight of a porphyrin unit) was necessary to kill 50% of the cells in the culture (IC50=18 μM), whereas for temoporfin the concentration necessary to attain the same toxicity for the same light dose was 0.2 μM (IC50=0.2 μM) (8).
The properties of porfimer sodium are inadequate for the penetration of biological barriers, namely the skin, because of its exceedingly high molecular weight, hydrophilicity and modest light absorption in the phototherapeutic window. Temoporfin partially resolves the issue of the molecular weight but it is exceedingly lipophilic for transdermal delivery and absorbs light just at the limit of the phototherapeutic window. The difficulty of these clinically approved photosensitizers to permeate the biological barriers, namely the skin barrier, is aggravated by the need for relatively large concentrations of these photosensitizers in the biological target to attain the phototoxicity required for PDT to offer a good therapeutic outcome.
It has not been appreciated in earlier uses of photosensitizers for PDT that the ideal properties of a photosensitizer for PDT could be combined in a single molecule with the ideal properties of drugs for topical applications. The ability to rapidly diffuse through biological barriers is critical for the success of, for example, intradermal or transdermal delivery of photosensitizers topically applied in the treatment of dermatological disorders, penetration of the photosensitizers in biofilms for the photoinactivation of bacteria, diffusion of the photosensitizer through nails for the treatment of fungal infections such as onychomycosis. The ability to rapidly diffuse through biological barriers is also critical for the rapid accumulation of the photosensitizer in its biological target, such as the permeation through the outer membrane of eukaryote cells or the membrane of bacterial cells. Such rapid diffusions shorten the time between the administration of the photosensitizer and the illumination of the target, which is advantageous in many applications of photodynamic therapy, and increase the phototoxicity towards the target. It would not be expected by the person skilled in the art that the carboxamide group in at least one meso position of the halogenated porphyrin derivatives shown in formula (I), in particular bacteriochlorins and chlorins, could contribute to the amphiphilicity and photostability of such bacteriochlorin or chlorin derivatives without compromising the generation of ROS, and with such a small contribution to the molecular weight of the photosensitizer that its diffusion through biological barriers is not impaired.
This invention discloses for the first time photosensitizers for PDT of hyperproliferative disorders and/or for the photoinactivation of bacteria or virus or fungi that meet all the criteria for the ideal photosensitizer and efficiently permeate biological barriers. The present invention also discloses processes to synthesize such photosensitizers and, by the way of examples, illustrates the use of these photosensitizers to kill cancer cells and inactivate bacteria. In a further embodiment of the present invention, the photosensitizers described herein are used for the theranostics of hyperproliferative tissues. Theranostics is a modality of image-guided therapy where the same compound is used to visualize the biological target and to obtain the desired therapeutic effect.